This application claims priority of the German patent application 101 39 920.0-42 which is incorporated by reference herein.
The invention concerns a scanning microscope and a method for scanning a specimen.
In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected or fluorescent light subsequently emitted by the specimen, laser beams usually being used for illumination. A specimen is scanned by means of a finely focused light beam. The focus of the illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device which generally comprises two tiltable mirrors that move in one specimen plane. The deflection axes are usually perpendicular to one another, so that one mirror deflects the incident beam in the X direction and the other in the Y direction. Tilting of the mirrors is achieved, for example, by means of galvanometer positioning elements.
With the galvanometer technology usually used at present, the maximally achievable scanning rates are limited, because of the inertia of the moving mechanical components, to a few hundred Hz for nonresonant galvanometers and a few kHz for resonant galvanometers. This ultimately results in relatively long measurement times for each sample.
Furthermore, the galvanometers are generally several centimeters long, the (usually round) mirrors having a diameter of approx. one centimeter. Beam deflection about two axes requires at least two galvanometer mirrors one behind another or nested in gimbal fashion within one another. This galvanometer assemblage requires a great deal of room in the microscope. It has therefore also already been proposed to use a beam deflection device having micromirrors for scanning a specimen; this considerably increases the scanning speed and scan quality.
DE 196 54 210 therefore furthermore proposes a particular embodiment of the scanning unit with which an incident beam can be deflected by mirrors in the X and Y directions and guided over the specimen to be examined. The scanning unit is constituted by three rotatable mirrors. The first and the second mirror are arranged in a fixed angular position with respect to one another, and are rotated together. The third mirror is rotatable independently of the first and second mirrors. With this arrangement it is possible to ensure a high image rate while at the same time eliminating serious imaging defects.
In confocal scanning microscopy specifically, the specimen to be examined is scanned in three dimensions with the focus of a light beam. A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the xe2x80x9cexcitation pinholexe2x80x9d), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels through the beam deflection device back to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors (usually photomultipliers) are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image.
A three-dimensional image is usually achieved by acquiring image data in layers. Scanning of the specimen in the axial direction (Z direction) is usually accomplished by so-called xe2x80x9cspecimen scanning,xe2x80x9d in which the specimen is moved in the Z direction using the specimen stage. It can also be performed, however, by displacing the objective in the axial direction, which is accompanied by a shift in the focus of the illuminating beam.
The reflected or fluorescent light emitted from the specimen upon illumination travels via a beam splitter and through a detection pinhole to a detector. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam, preferably at fixed time intervals. The specimen can thus be scanned in three dimensions one grid point at a time, and at each scan point a reading that is representative of that specimen point can be ascertained.
The lasers usually used as illumination sources in scanning microscopes allow optimum illumination of the specimen in order to obtain the desired result. One important aspect regarding the quality of a scanning microscope is precise imaging of the rotation point of the beam deflection device into the pupil of the objective being used. In the case of beam deflection devices that, because of their geometry, have more than one rotation point, adaptation of the imaging in terms of image quality should be optimally adjustable. But because the location of the pupil depends on the one hand substantially on the objective being used and on the other hand on the wavelength of the illuminating beam, known microscopes do not offer a satisfactory solution to this problem. The illuminating beam is therefore mostly not tilted in the Pupil of the objective which leads to unwanted aberrations cause of non telecentric imaging.
It is therefore the object of the present invention to propose a scanning microscope which avoids the mentioned aberrations especially at different wavelength.
According to the present invention, this object is achieved by a scanning microscope comprising:
an illumination source for generating an illuminating beam;
a beam deflection device that defines at least one illuminating beam rotation point for deflecting the illuminating beam,
a means for imaging the illuminating beam rotation point,
an objective that defines at least one pupil,
a device for axial displacement of an image of the illuminating beam rotation point into the at least one pupil of the objective.
It is a further object if the invention to propose a method, which allows to scan a specimen especially at different wavelength and which avoids the problems of non telecentric imaging.
This object is achieved by a method for scanning a specimen with a scanning microscope comprising the steps of:
Generating an illuminating beam with an illumination source,
Transferring the illuminating beam to a beam deflection device that defines at least one illuminating beam rotation point,
Imaging an image of the illuminating beam rotation point,
Focusing the illuminating beam onto the specimen with an objective that defines at least one pupil,
Displacing the image of the illuminating beam rotation point into the at least one pupil of the objective with a device for axial displacement of the image of the illuminating beam rotation point.
The scanning microscope according to the present invention thus comprises an illumination source with which the illuminating radiation necessary in the scanning microscope is generated. Advantageously, a laser is used to generate the illuminating beam. With the beam deflection device present in the scanning microscope, it is possible to deflect the illuminating beam line by line, i.e. in the X and Y directions, so that the specimen to be examined can be scanned. This beam deflection device defines, at the deflection points, an illuminating beam rotation point. The illuminating beam rotation point is the virtual intersection point of the deflected illuminating beams. In order to influence the imaging of this illuminating beam rotation point, a device for axial displacement of an image of said illuminating beam rotation point into the pupil of the object is provided. With the use of the device according to the present invention for axial displacement, it is possible to adapt the location of the image of the illuminating beam rotation point to the particular objective and illuminating beam wavelength being used. This adaptation results in substantially perpendicular imaging of the illuminating light beam through the objective onto the specimen, since imaging of the illuminating beam rotation point into the pupil of the objective guarantees that even with line-by-line scanning, the illuminating beam is directed substantially perpendicularly onto the specimen at every point. In addition, with the microscope according to the present invention it is possible to use different illuminating wavelengths, since the difference in location (already described above) of the pupil at different illuminating beam wavelengths can be compensated for with the scanning microscope according to the present invention.
In a preferred embodiment of the invention, the device for axial displacement of the image of the illuminating beam rotation point is coupled to the beam deflection device itself. Advantageously, the beam deflection device is combined for that purpose into a module which is equipped with the aid of a manually adjustable mechanism or in motorized fashion can be modified in terms of its axial location in such a way that the image of the illuminating beam rotation point can be placed in the pupil of the objective. The device for axial displacement of the image of the illuminating beam rotation point is in a preferably embodied in such a way that a substantially continuous or stepwise displacement of the components to be displaced of up to 30 mm from their basic position is possible.
In a further preferred embodiment, however, lenses present in the microscope that are arranged in front of the objective and modify the focal length can also be equipped with a mechanism that modifies their axial position. With this as well, the respective lenses or even an entire lens system can be positioned in such a way that the image of the illuminating beam rotation point is imaged in the pupil of the objective. The properties of the particular objective and illuminating beam wavelength being used are taken into account in this context.
In a further embodiment, the entire scanning unit which substantially contains the illumination and detection pinholes, the main beam splitter, the beam deflection device, the detector, and certain optical systems for shaping and guiding the light beamsxe2x80x94can be displaced in such a way that the image of the illuminating light beam rotation point is imaged into the pupil of the objective (or at least into the vicinity of the pupil).
To allow this adjustment operation to be performed conveniently and easily, the pupil locations for each of the objectives and for the illuminating wavelengths used in the microscope must each be known. These can, for example, be determined in a previous working step for the various objectives and wavelengths used, and stored in a memory unit in the microscope. Advantageously, however, the manufacturer""s data are used. It is thereby possible to automate the operation of adjusting the axial displacement. For automatic adjustment, it is necessary merely to know the objective being utilized and the illuminating beam wavelength that is in use. From the data stored for the specific combination of objective and illuminating beam wavelength, the particular position required for the beam deflection device, scanning unit, or axially displaceable lenses can then be ascertained. A displacement travel is then determined by comparing the present position to the setpoint position, and the device for axial displacement is then activated so that the desired displacement occurs.
With the scanning microscope according to the present invention and the method according to the present invention for scanning a specimen it is thus possible to make a contribution toward automating the adjustment operation; this on the one hand reduces the error rate and on the other hand shortens the setup time prior to operational readiness of the microscope.